Project Summary/Abstract The focus of this proposal is to understand how a replisome responsible for DNA replication is assembled from its component proteins, how it functions in the coordination of leading- and lagging-strand DNA synthesis, and how it responds to a damaged DNA template. The systems being examined to gain such insights are the bacteriophage T4 replisome derived from eight proteins and the collection of human proteins/enzymes responsible for replicative DNA synthesis and translesion synthesis. The T4 proteins can be grouped into subassemblies: the holoenzyme and the primosome reconstituted from the polymerase and clamp proteins, and from the helicase and primase proteins, respectively. We are specifically interested in how these T4 subassemblies carry out their functions such as lagging- strand polymerase recycling; the disassembly of the lagging-strand holoenzyme; and their relative orientation within the replisome, in particular the two holoenzymes. The ultimate goal is to integrate these finding with the observation of a functioning T4 replisome carrying out leading- and lagging-strand synthesis at the single-molecule level. Answers to these complex questions will be sought with a wide assortment of techniques applied to single-molecule and molecular ensembles. The human replisome has the added complexity of using two different replicative polymerases for leading- and lagging-strand DNA synthesis. We will use biochemical studies to characterize the formation and stability of the replicative holoenzyme complexes. We will extend in parallel our methodology to how a replisome copes with the problem of a damaged template base in creating a complementary strand. DNA damage tolerance pathways include template switching and translesion synthesis in euckaryotes Of particular interest is how a lesion is recognized, what is the signal for and rol of PCNA ubiquitination in the switch of a Y-family polymerase for the replicative polymerase; the composition of the translesion synthesis holoenzyme complex; and ultimately the pathway for reversal and restoration of the replicative holoenzyme. Answers to these questions will be sought by reconstituting translesion synthesis with the human proteins and enzymes.